KR20150143625A - Medical use of syndecan-2 - Google Patents

Abstract

A composition comprising SDC2, SDC2, a vector encoding SDC2, and a compound modulating the expression of SDC2 by the cell are used for the treatment of mammals, such as human cells, to achieve immunomodulation or other specific therapeutic intervention do. The cells are treated by binding the cells to SDC2, treating the cells with an antibody or fragment thereof that binds SDC2, or modulating the expression or activity of SDC2 by the cells. The cells or tissues are derived from cells that have been treated for therapeutic use based on their immunomodulatory or other therapeutic properties.

Description

MEDICAL USE OF SYNDECEN-2 < RTI ID = 0.0 >

The present invention relates to immunomodulatory and therapeutic agents and compositions, and uses thereof. The present invention also relates to testing and modifying existing cell-based products to influence immunomodulation and / or other therapeutic properties.

A number of mesenchymal stem cell (MSC) -related products for clinical use are known and more products are under development and are expected to be approved for future human use. MSCs exhibit some immunomodulatory properties that can contribute to its therapeutic value, and MSCs are particularly used for the treatment of autoimmune diseases. The immunosuppressive potential of human MSCs is evidenced by studies showing inhibitory effects on the proliferation of, for example, T-cells, B-cells, dendritic cells and natural killer cells (Han et al., 2012).

EP 1795588 discloses the use of adipose tissue-derived MSCs for the treatment of graft-versus-host disease (GVHD); The cells are said to exhibit immunosuppressive properties.

EP 2545928 relates to MSC-containing cell preparations having immunosuppressive ability maintained by serum-free or low serum cultures.

WO < RTI ID = 0.0 > 2012/111997 < / RTI > discloses a combination therapy that provides MSCs together with immunoregulatory T cells to achieve immunosuppression suitable for MSC-based therapies.

WO 2009/1105624 discloses compositions and methods relating to the delivery of molecules and polypeptides together into cells to improve the therapeutic efficacy of such molecules. The delivery of the growth factors may involve contacting the growth factors with their receptor and co-receptor; For example, with sindecane 1, 2, 3 and 4. Co-transfer of sindecane and growth factor protects the growth factor from proteolysis, increases their activity, and targets the growth factor to the cell surface to promote growth factor signaling.

KR1020100106744 relates to a composition for preventing or treating migratory dermatitis or melanin formation-related diseases. The composition for the prevention or treatment of migratory dermatitis contains syndecan-2 as the active ingredient. The migratory dermatitis diseases considered are melanoma, hyperpigmentation, hypopigmentation or vitiligo.

WO 02/087609 discloses a method of preventing a papillomavirus infection in a mammal comprising a soluble peptide, protein, or fusion protein that binds to the papillomavirus particle as a ligand to a cedecane with a heparan sulfate glycose aminoglycan chain attached thereto ≪ / RTI >

WO 03/062386 relates to methods and materials relating to modulating sindecane levels and angiogenesis in an animal. Discloses a nucleic acid encoding a sindecane polypeptide and a sindecane polypeptide. These are used to generate polynucleotides and polynucleotide analogs for angiogenesis regulation. Methods for identifying sindecane- and angiogenesis-modulating agents are also discussed.

Mukhopadhyay, A. et al., J. Trauma Injury Infect. Crit. Care, 2010, vol. 68, pp. 999-1008] assume that sindecane-2 and FGF-2 interact with each other to produce divergent-2 divergence from cells and in turn activate events that are the cause of the keloid phenotype.

Kaur, C. et al., Glia, 2009, vol. 57, pp. 363-349] examines the expression of sindecane-2 in amoeba-shaped microglia and indicates that the expression is upregulated by hypoxia.

Contreras, H. R. et al., Biochem. Biophys. Res. Comm., 2001, vol. 286, pp. 742-751 examines the expression of sindecane-2 in cancer and indicates that sindecane-2 expression is involved in the differentiation of some cells into a migrating mesenchymal-like phenotype.

Sojoong Choi, et al., Biochem. Biophys. Res. Comm., 2012, vol. 417, pp. 1260-1264] examined the role of the substrate metalloproteinase-7 (MMP-7) on the release of sindecane-2 from colon cancer cells. It was observed that MMP-7 directly mediates the divergence of sindecane-2 from the cells.

Alexopoulou, A. N. et al., BMC Cell Biol., Vol. 9, 2008, doi: 10: 10.1186 / 1471-2121-9-2] investigated vectors useful for long-term expression of transgene during mesenchymal stem cell differentiation. One of the vectors used above encodes syndecan-2.

WO 2011/153458 discloses a method of preventing dengue fever infection, including interfering with Dengue virus binding to sindecane present on cells targeted by Dengue virus. Also disclosed are pharmaceutical compositions related to the above objects.

Mytilinaiou, M. et al., IUBMB Life, 2013, vol. 65, pp. 134-143] examined the role of sindecane-2 as a modulator of cell adhesion to fibrosarcoma cells mediated by TGFβ2 / Smad2.

Kim, Y. et al., Oncogene (2003), vol. 22, pp. 826-830] observed that reduced sindecane-2 expression correlated with changes in trichostatin-A-induced morphology and decreased tumorigenic activity in colorectal carcinoma cells. In addition, down-regulation of the expression of syndecan-2 by antisense cDNA mimicked this morphological change, thereby reducing the anchor-independent growth of colon cancer cells.

Chung, H. et al., Journal of Biological Chemistry, vol. 287, no. 23, pp. 19326-19335, 2012] found that the melanocortin 1 receptor modulates melanoma cell migration by modulating sindecane-2 expression. This is because the melanocortin 1 receptor inhibits the activation of p38 MAPK and subsequently increases sindecane-2 expression and migration in melanoma cells.

Peterfia, B. et al., PLoS ONE, vol. 7, no. 6, e39474] observed that sindecane-1 induces the malignancy of mesenchymal tumor cell lines through the induction of sindecane-2 expression. Thus, syndecan-1 works in conjunction with syndecan-2 to increase the proliferation, migration and metastasis of human fibrosarcoma cell lines.

Ruiz, X. et al., PLoS ONE, vol. 7, no. 8, e43049] discloses that syndecan-2 is a novel target for insulin-like growth factor binding protein-3 (IGFBP-3) and is overexpressed in syndecan-2 fibrosis. The increased SDC2 expression is due, at least in part, to the activity of two fibrogenic precursors, TFG [beta] and IGFBP-3.

Dieudonne, F-X et al., Journal of Bone and Mineral Research, vol. 27, no. 10, pp. 2118-2129] discloses how sindecane-2 expression is upregulated by doxorubicin in osteosarcoma cells. T-cell factor (TCF) is responsible for the inhibition of syndecan-2. Thus, targeted inhibition of TCF activity promotes sensitization to sindecane-2 expression and doxorubicin in osteosarcoma cells and bone tumors in mice.

KR 20120013915 discloses diagnostic compositions of colon cancer containing an agent for measuring the level of expression of a sindecane-2 peptide fragment. The agonist contains an antibody specific for the sindecan-2 peptide fragment.

Huang, X. et al., Oncology Reports 2009, vol. 21, pp. 1123-1129] discuss the prognosis of altered expression of sindecane-2 (SDC2) and cysteine-rich 61 (CYR61) in esophageal squamous cell carcinoma.

A common problem in the art is that there is insufficient immunosuppression for the MSC-based product used alone, or that the immunosuppressive properties of the MSC are lost, i.e., are not maintained over time in the cell passaged culture . It is desirable to be able to maintain, stimulate or restore this property.

Immunosuppressive regimens can be used to prevent or reduce graft rejection, and clinically used immunosuppressive regimens typically include combinations of multiple agents currently used. It may be desirable to identify alternative immunosuppressants and therapies. Similarly, it may be desirable to identify agents and treatments that promote angiogenesis. In addition, it may be desirable to be able to analyze the efficacy of potential therapeutic products in the art. The proposed assay is based on soluble TNFR1 but requires an alternative, preferably an improved assay.

Object of the invention

It is an object of the present invention to provide alternative agents and treatments that are immunomodulatory and / or have other therapeutic properties as specified in the present invention. It is an object of certain embodiments of the present invention to provide agents and treatments for use in making products or compositions comprising cells or tissues less immunogenic or even more immunosuppressive.

SUMMARY OF THE INVENTION

In the use of embodiments of the present invention, increased syndecan-2 (SDC2) expression and / or activity has been shown to enhance immunotherapy and / or have other therapeutic properties. Accordingly, the present invention provides a composition comprising SDC2, SDC2, a vector encoding SDC2, and a compound that modulates the expression of SDC2 for use in the treatment of cells, wherein said cells are for therapeutic use. These compositions, vectors, and compounds may themselves be used in therapeutic treatments, particularly in human therapeutic treatments, such as immunomodulating or other therapeutic uses as described herein.

The present invention also encompasses a method of treating a cell population comprising contacting the cell with SDC2, treating the cell with an antibody or fragment thereof that binds SDC2, and / or modulating the expression or activity of SDC2 by the cell or cells Of the present invention. The cells produced by the above methods and also the cells and tissues derived from the treated cell rotors form another part of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention include compositions comprising SDC2, methods using SDC2, and specific uses of SDC2. Suitably, said SDC2 is in soluble form and in a particular embodiment said is human or horses SDC2 but is generally derived from SDC from mammals, in particular from human, mouse, rat, dog, horse, rabbit, sheep, SDC2 is included in the present invention.

The composition may comprise pharmaceutically acceptable excipients and / or carriers suitable for use in the medicament. The composition may comprise a cell culture medium (e.g., SDC2 in a cell culture medium) suitable for use in cell culture, for example, for addition to a cell cultured in the manufacture of a therapeutic cell-based product, Can be formulated as a medium. The composition can be formulated to slow release and optionally modified to improve half-life. For example, the SDC2 (or fragments, variants, derivatives or analogs) can be PEGylated or PSA modified. The composition may be formulated for injectable use, for example, in injection water or saline solution. The composition may be formulated for inhalation, for example by spraying. The SDC2 may be produced in recombinant form or may be isolated from cells expressing or overexpressing SDC2. In the tested example, SDC2 was isolated from the supernatant of cells expressing SDC2 and recombinantly prepared; As indicated elsewhere in the present invention, SDC2 can be diverted into the culture medium and thus SDC2 can be harvested from a culture of SDC2 expressing cells.

The present invention also provides SDC2; Again preferably, an expression vector encoding human or horse SDC2 is provided, but in general also a vector encoding SDC2 from the mammal mentioned forms an embodiment of the invention. The term "expression vector" refers to a nucleic acid, e. G., Linear or cyclic DNA or RNA, containing an SDC2 encoding segment operatively linked to additional segments provided for transcription. Such additional segments may include a promoter (many of which are known, suitable examples include the CMV and UbC promoters), terminators and UTR sequences, and may contain one or more replication origin, one or more selectable markers, A decylation signal, and the like. The expression vector may generally be derived from plasmid or viral DNA, or may contain both of these elements. One suitable vector of the invention, as used in the following examples, is an adenoviral vector. Other suitable viral vectors include retroviruses, lentiviruses, and adeno-associated viruses. A preferred vector for the expression of human SDC2 comprises the coding sequence from SEQ ID NO: 1 or 2 (e.g., no poly A tail), or an allelic variant thereof, or another nucleotide sequence encoding SEQ ID NO: 3 or 4.

SDC2 may be excreted by the cells into the culture medium and such ectodomain release is a highly regulated direct action of the enzyme generally referred to as sheddase. Substrate metalloproteinases (MMPs) are known as shaders of sindecane. In particular, MMP-7 has been shown to be necessary for SDC2 divergence (Ryu et al., 2007). However, it has been reported that growth factors (Subramanion et al., 1997), chemokines (Li et al., 2002; Brule et al., 2006; Charnaux et al., 2005), heparanase (Yang et al., 2007) Various extracellular stimuli including cell stress (Fitzgerald et al., 2000) have also been shown to induce divergence of SDC2.

In a particular embodiment of the invention described in more detail below, SDC2 diverted into the culture medium from the surface of human MSCs had immunosuppressive properties when the SDC2-containing medium was used for the treatment of separate cell populations. Thus, the present invention provides the use of SDC2 to provide immunosuppression which is the therapeutic effect. Treatment, e. G., Cells for transplantation may be treated in this manner, e. G. By exposure to SDC2, to reduce the immunogenic properties of the cells. The immunosuppressive effect achieved in accordance with the present invention may also be used in combination with other cell-based therapies to reduce or delay the risk or occurrence of an adverse immune response in a patient, for example. Thus, the SDC2 and SDC2-containing compositions of the present invention, as well as the vectors and compounds of the present invention, can be used in combination with other therapeutic compositions, such as cell therapy products, that can be or comprise MSCs and / It can be administered to patients.

Compounds that modulate the expression of SDC2 by cells for use in the treatment of cells are additionally provided by the present invention. In an embodiment of the invention, the compound increases the expression of SDC2. Examples of such compounds include compounds that activate p53, compounds that activate ERK, p38 or JNK SAPK kinase pathways, compounds that activate hypoxia inducible factor (HIF) -mediated pathways, TGF, BMP, Lefty, Compounds that agonize the Nodal and Activin pathways, compounds that function the NOTCH pathway such as Jagged1, Jagged2, DLL1, DLL2, DLL3 and DII4, Hedgehog ) Compounds that function in the signaling pathway, compounds that function the WNT pathway, compounds that activate the androgen receptor or estrogen receptor pathway, and compounds that activate the NFkB pathway. Examples of compounds for use in augmenting SDC2 expression include but are not limited to p53 regulated chemotherapeutic agents (e.g., nutrin, 5-FU, doxorubicin, cisplatin, gemcitabine, taxol), HDAC inhibitors, PPAR agonists, DMOG ), Troglitazone (PPAR alpha and PPAR gamma), phosholin, colosin (a water soluble version of the phoscholine, cAMP stimulant), WY4,643 (pyridinium acid, peroxisome proliferator activated receptor- (HDAC inhibitor), valproic acid (HDAC inhibitor), or SAHA (another HDAC inhibitor), spliceomicin, Sir2 (tribal HDAC inhibitor), tricostatin A ). Thus, expression enhancement of SDC2 in cell therapy products can be used to render the product immunosuppressive or to increase the immunosuppressive properties of the product.

The problem addressed in the present invention is the provision of yet another immunomodulatory and / or other therapeutic intervention. In the use of the present invention, the above compositions, vectors and compounds can be used for therapeutic treatment.

For example, in a series of embodiments of the invention, the composition, vector or compound is for use in a therapeutic treatment comprising immunomodulation. Increasing the amount and / or expression and / or activity of SDC2 promotes immunosuppression; The present invention thus provides a therapeutic treatment comprising immunosuppression.

The compositions of the invention, as well as the vectors and compounds of the present invention, alone or in combination therapies as referred to in the present invention, can be used to treat pulmonary diseases such as ARDS, COPD and IPF. Intravenous injected stromal cells tend to accumulate in the lungs during the test, indicating that delivery to the lungs can be achieved. SDC2 can also be sprayed for pulmonary delivery.

The compositions and treatments of the present invention that enhance SDC expression and / or activity and also the corresponding vectors and compounds of the invention are generally suitable for any indication that requires immunosuppression or immunosuppression may be beneficial; These indications are generally suitable for graft transplantation, psoriasis, indications with asthma and autoimmune components, such as allergies, colitis, dermatitis and inflammatory diseases.

In embodiments, the compositions and treatments of the invention and also the vectors and compounds of the invention may be formulated with anti-inflammatory agents, such as anti-TNF antibodies (examples of such antibodies include infliximab (Remicade), AdalmyMap (Humira) (Including heartworms), Toli mugpegol (simia), and Goli Mumap (symphony), or circulating receptor fusion proteins such as etanercept (Enbrel). The present invention may be used with anti-CD3 antibodies, examples of such antibodies include Mylomopa-CD3, Otellicus Map, Tepripulmag, and Non-Sylic Map. The combination may be used for immunosuppressive therapy; Which can be used for the treatment or prevention of transplant rejection and other inflammatory or autoimmune diseases such as Crohn's disease, ulcerative colitis and type 1 diabetes, and induction of immune tolerance.

In a particular embodiment of the invention described in more detail below, increased SDC2 on the surface of human MSCs makes the cells more immunosuppressive compared to control MSCs. Thus, the present invention provides the above therapeutic effect by providing increased cell expression or activity or amount of SDC2. The transplantable cells may be treated in this manner to reduce the immunogenic properties of the cells. The immunosuppressive effect achieved according to the invention may also be used in combination with other cell-based therapies, for example to reduce or delay the risk or occurrence of an adverse immune response in a patient. The treated cells of the invention may be administered to a patient in conjunction with another therapeutic composition, such as a cell therapy product, that may or may be MSC and / or stromal stem cells.

The cells of the invention may be administered prior to, concurrent with, or after the other therapeutic product. The cells of the present invention may be administered beforehand to support tolerance of the patient ' s immune system to subsequent administration of cell therapy products, e. G., Implants.

The compositions of the present invention, as well as the vectors and compounds of the present invention, as well as compositions, uses, methods and treatments of the present invention that enhance SDC expression and / or activity, can be used to treat cancer. Cancer, specifically, hepatocellular carcinoma, cervical cancer, pancreatic cancer, prostate cancer, breast cancer, colon cancer, fibrosarcoma, hydroblastoma, and astrocytoma can generally be treated using the present invention.

In the embodiments of the present invention, the treated cancer is a solid tumor. Moreover, embodiments of the present invention include targeting the treatment to a cancerous substrate that is considered to be essential for sustaining cancer growth and growth, advantageously focusing the cancerous treatment on the cancerous substrate.

In a particular embodiment of the invention described in more detail below, SDC2 has an anticancer effect against pancreatic cancer, prostate cancer, breast cancer and colon cancer, respectively.

The compositions of the present invention, as well as the vectors and compounds of the present invention, as well as the compositions, uses, methods and treatments of the present invention that enhance SDC expression and / or activity, can be used to treat inflammatory and / or inflammatory diseases. Thus, anti-inflammatory effects can be achieved using the present invention.

In certain embodiments of the invention described in more detail below, SDC2 exhibited an antiinflammatory effect upon analysis.

The compositions of the present invention, as well as the vectors and compounds of the present invention, as well as the compositions, uses, methods and treatments of the present invention that enhance SDC expression and / or activity are used for wound healing or bone healing, It can be used for promotion.

In a particular embodiment of the invention described in more detail below, the cells expressing SDC2 significantly increased wound healing in one model. In analogy to other data of the present invention, it supports a similar activity of SDC2 in wound healing, including wound healing and fracture healing.

The compositions of the present invention, as well as the vectors and compounds of the present invention and also the compositions, uses, methods and treatments of the present invention that enhance SDC expression and / or activity can be used to treat a variety of endocrine disorders, , And exercise-induced pulmonary hemorrhage (EIPH) (also known as "bleeding" or "bleeding episodes").

Also provided by the present invention is a pharmaceutical composition for treating an animal, particularly a mammal and, for example, a human or horse disease or disorder. The pharmaceutical composition suitably comprises a compound, composition or vector of the present invention in an amount effective to treat the disease or disorder in the animal. Thus, the active agent of the present invention may be administered together with an acceptable pharmaceutical carrier. For example, the active agent can be administered in a pharmaceutically acceptable liquid medium for injection. An example of a liquid medium is saline, phosphate buffered saline, which also optionally contains additional substances such as dimethyl sulfoxide (DMSO) and human serum albumin. Methods of administering therapeutic proteins and peptides are known in the art. Preferably a human, comprising administering to the animal, preferably a human, an active agent of the invention.

The pharmaceutical compositions disclosed herein are useful for medical and veterinary uses and may be administered to an individual alone or in combination with other supplemental active ingredients, agents, drugs or hormones. The composition may optionally comprise a pharmaceutically acceptable carrier, for example, a pharmaceutically acceptable vehicle, stabilizer, diluent, excipient, adjuvant or excipient. Useful pharmaceutically acceptable carriers include, but are not limited to, aqueous media such as water, saline, glycine, hyaluronic acid, and the like; Solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like; menstruum; A dispersion medium; Coating agents; Antimicrobial agents and antifungal agents; Isotonic and absorption delaying agents; Or any other inert component. The choice of the pharmaceutically acceptable carrier may vary depending on the mode of administration. Except insofar as any of the pharmacologically acceptable carriers is contra-indicated with the active ingredient, use thereof in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in the following references: Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., Eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. Goodman & Gilman ' s The Pharmacological Basis of Therapeutics (Joel G. Hardman et al., Eds., McGraw-Hill Professional, 10th ed. and Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications, 4th edition 2003).

The pharmaceutical compositions disclosed herein may also contain other pharmaceutically acceptable ingredients such as, but not limited to, buffers, preservatives, tonicity adjusting agents, salts, antioxidants, osmolality adjusting agents, physiological materials, pharmacological agents, bulking agents, emulsifiers , Wetting agents, sweetening or flavoring agents, and the like. Various buffers and means for pH adjustment may be used to prepare the pharmaceutical compositions disclosed herein, provided that the resulting formulation is pharmaceutically acceptable.

A variety of routes of administration may be useful for administration of the therapeutic compounds disclosed herein. A topical, enteral or parenteral route of administration may be suitable as such and such routes include both topical and systemic delivery of the therapeutic compounds or compositions disclosed herein. Compositions for inhalation, topical, intranasal, sublingual, injection, infusion, drip, rectal and / or vaginal use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions.

Separately or together, an increasing amount of SDC2 and / or expression and / or activity promotes angiogenesis; Thus, such an embodiment of the present invention provides a therapeutic treatment that includes or promotes angiogenesis.

The present invention provides a method of treating such cell populations comprising binding cells with SDC2. The method can be accomplished by combining the cell with (i) a cell expressing SDC2, or (ii) a composition or compound according to other embodiments of the invention. In vitro and in vitro treatment.

In another embodiment, the invention can be adapted to reduce the amount of SDC2 and / or expression and / or activity to provide therapeutic treatment including immunostimulation and / or immunostimulation.

The cells to be treated may be stem cells such as stromal stem cells or MSC and are suitably mammalian cells, preferably human, mouse, rat, dog, horse, rabbit, sheep, And human cells.

The present invention further provides a method of treating such cell populations, comprising modulating the expression or activity of SDC2 by the cells or cells in the population. Again, in vitro and in vitro treatment are included. Regulation of the expression or activity of the SDC2 can modulate the immunosuppressive properties of the cell. The modulation may also increase the expression of SDC2 to increase another immunosuppressive property of the cell. The modulation may increase the expression of SDC2 to increase another therapeutic characteristic of the cell.

To treat cells, the cells may be treated with a compound that promotes the expression of SDC2; An example is provided elsewhere in the present invention. The cells can be transfected with a vector encoding SDC2.

The cells may be treated to increase exogenous SDC2 expression of the cells, for example, by exposure to culture or environmental conditions that increase SDC2 expression. Multiple strategies can be used to stimulate SDC2 expression, particularly in stem cells, including stem cells and MSCs. These include p53 activation by specific biological factors, compounds or stresses; Compounds that activate the ERK, p38 or JNK SAPK kinase pathway, biological factors and stress; Biological factors, compounds or stresses that activate a hypoxia-inducible factor (HIF) -mediated pathway; Biological agents, compounds or stresses that function TGF, BMP, Lefty, nodal and actibin pathways; Compounds, or stresses, such as Jagged1, Jagged2, DLL1, DLL2, DLL3, and DII4; Biological agents, compounds or stresses that functionalize the hedgehog signaling pathway; Biological agents, compounds or stresses that function the WNT pathway; Biological agents, compounds or stresses that activate the androgen receptor or estrogen receptor pathway; And biological factors, compounds or stresses that activate the NFkB pathway.

In embodiments of the invention, SDC2 expression was increased by direct or indirect activation of the tumor suppressor protein p53. Without wishing to be bound by theory, this may be due to the presence of "putative" p53-binding sites in the promoter of the SDC2 gene (e. Such activation can be accomplished in a number of ways, some of which, for example, HDAC inhibition, growth arrest, starvation, toxin exposure and / or chemotherapy are known to the skilled artisan.

Serum starvation (SS) and growth arrest (fusion) all stimulated SDC2 expression. SS and growth arrest are both known p53 stimuli.

HDAC inhibitors and sirtuin can be used to increase SDC2 expression. In the use of the present invention, splittomycin, valproic acid, and 2-pyrrolidinone-n-butyric acid (PBA) all increased the level of SDC2 RNA at 24 hours, and by the class III HDAC inhibitor splittomycin There was a firm stimulus. The sirtuin class of HDAC, including SIRT1 and SIRT2, can be used to increase SDC2 expression. The sirtuin is an NAD-dependent HDAC and is reported to deacetylate the p53 protein, i.e. the acetylation of p53 is reported to be important for its transcriptional activation / transcriptional activity. Thus, sirtuin is a key regulator of p53 activity. Examples of sirtuin include nicotinamide, sirtinol, EX-527 and tenovin. In another test of the invention, solvent DMSO also increased SDC2 RNA levels. DMSO is also reported to contain HDAC inhibitor (p53 activation) capability.

Induction or induction of hypoxia in the cells can be used to increase SDC2 expression; In another series of tests, the hypoxic imitation DMOG (dimethyl oxaloylglycine) produced a very firm increase in SDC2 RNA. DMOG is a hypoxia imitation material, and other hypoxia imitation materials for use in the present invention include cobalt chloride, EDHB and desperoxamine. Hypoxia is reported to stimulate p53 (via ATR / CHK1 kinase).

Toxins such as the environmental toxin benzo [a] pyrene-7,8-diol-9,10-epoxide (BPDE) can be used to increase SDC2 expression. Other p53 activators for use in the present invention include the use of gamma-irradiation, MDM2 inhibitors (nutrilins) and chemotherapeutics such as cisplatin and gemcitabine.

Thus, embodiments of the present invention include treatment of cells that activate p53 to increase SDC2 expression and / or divergence.

The cell to be treated is suitably a mammalian cell, preferably a human, mouse, rat, dog, horse, rabbit, sheep, cow or pig and especially a horse and a human cell. Prior to treatment, the expression of SDC2 by the cells may be low or absent, and thus the treatment provides a level of SDC2 that was not previously seen for the cells. On the one hand, the level of expression prior to treatment may be initially high but decreased over time, and the treatment is to restore the pre-existing SDC2 expression level, for example, immunosuppression that has been lost over time from the cell or tissue And / or to restore other therapeutic properties.

Regulation of expression or activity of SDC2 may reduce the amount and / or activity and / or expression of SDC2 in order to reduce the immunosuppressive properties of said cells. The modulation may reduce the amount and / or activity and / or expression of SDC2 to reduce the angiogenic properties of the cell. Further embodiments of the invention include treating the cells to induce immunostimulation and / or immunostimulation. Cells can be treated to remove native SDC2 expression; For example, antisense therapy or other therapies may be used to knock-out or rust-down SDC expression.

All methods of the present invention further comprise deriving offspring cells or tissues from the treated cells; The invention also extends to cells or tissues obtained from the methods.

Thus, in an embodiment of the present invention, the level of SDC2 on the cell surface and the cellular medium is related to immunomodulation and / or other therapeutic properties. Furthermore, the present invention provides a test method comprising testing a cell therapy product, which may comprise cells and / or tissues, against SDC2. This can be accomplished using assays based on binding to antibodies to SDC2, preferably SDC2-specific antibodies. Conventional SDC2 ELISA methods may be suitable. SDC2 can be measured on the cell surface or in media or both. The level of SDC2 can be compared to a pre-measured standard, where elevated levels indicate the presence of immunosuppressive and / or other therapeutic properties, and reduced levels indicate absence thereof.

In an embodiment of the present invention, an assay for the efficacy of the product, including assaying the cell for expression of SDC2 or analyzing the presence of a therapeutic product (e. G., A solution) comprising the cell against the presence of SDC2 . Another embodiment of the present invention is the use of SCD2 levels in an assay for the efficacy of cell therapy products.

In general, in the use of the test / analysis method of the present invention, ELISA can be used to monitor the efficacy of SDC2 divergence and thus the specific placement of human MSCs.

Cells or products failing the test / analysis may be discarded or treated to increase their SDC2 expression. The analysis of SDC2 can be used prior to determining whether to treat the cells or tissues according to other methods of the present invention.

The analysis protocol of the present invention

1. Analyze SDC2 levels in potential products;

2. comparing the level to a pre-measured minimum value for an acceptable therapeutic product,

a. If the level is greater than or equal to the minimum value, the potential product is allowed;

b. If the level is below the minimum value, the product is not allowed;

3. optionally, then comparing said level to a pre-measured maximum value for an unacceptable therapeutic product;

a. The product is rejected if the level is below the maximum value;

b. If the level is intermediate between a minimum value for an acceptable therapeutic product and a maximum value for an unacceptable therapeutic product, the potential product may be processed to increase its SDC level, and then repeat the analysis

.

The aforementioned pre-measured minimum and maximum values for the cut-off SDC2 level for the assay may vary depending on other tests, the cell used, the nature of the treatment, and other factors. The pre-measured minimum level for the therapeutic product may be SDC2 levels of approximately 1200 pg / ml, 1300 pg / ml, 1400 pg / ml, 1500 pg / ml or more. The maximum pre-measured levels for unacceptable agents may be about 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml or 600 pg / ml; The level may be higher or lower than these described values, but will generally be significantly lower than a pre-measured minimum value for an acceptable product. On the one hand, any formulation below a premeasured minimum value for an acceptable therapeutic product may not be acceptable but may be suitable for treatment to raise the SDC2 level to an acceptable level.

In certain embodiments of the invention described in more detail below, poor MSC donor cell preparations failed the SDC2 assay while acceptable donor formulations failed.

Thus, the present invention provides "batch release" or quality control efficacy analysis for stem cell and other cell therapy patches or products prior to use prior to use, shipping, and the like.

Sindecan-2 (SDC2) (also called pibroglycan and now CD362) is a transmembrane (type I) heparan sulfate proteoglycan and is a member of the syndecane proteoglycan family. Reference to SDC2 in the present invention refers generally to all species, preferably humans, mice, rats, dogs, horses, rabbits, sheep, cows and pigs, especially horses and more preferably human, Quot;

The invention also relates to a pharmaceutical composition comprising an antibody against SDC2, a therapeutic method using an antibody against SDC2, the use of an antibody against SDC2, and an antibody against SDC2. At least the anti-syndecan 2 antibody, orb13481, which is reactive with human, mouse and rat, is available from Biorbyt Ltd. (UK CB25 9QR Cambridge Water Beach, Denny Industrial Center, Pembroke Avenue 12). Additional SDC2 antibodies, Catalog # MAB29651 (clone 305507), which is reactive with human, mouse, rat, horse, rabbit and pig, are available from R & D Systems Inc. Antibodies to SDC2 fragments as disclosed herein are also encompassed within the term anti-SDC2 antibody.

The data from the use examples of SDC2 demonstrate the role of SDC2 as a predominant negative inhibitor of the activity of native SDC2. Thus, the present invention provides the therapeutic use of antibodies against SDC2 antagonists, such as SDC2 or SDC2, corresponding to the use of SDC2 disclosed in the present invention and SDC2 positive cells disclosed in WO 2013/117761. The present invention thus provides antagonists (e. G., Antibodies) to SDC2 for use in immunosuppression, treatment of inflammation, treatment of cancer and wound / bone healing.

Thus, the present invention also provides an antibody against SDC2 for use as a tumor suppressor. Antibodies to SDC2 may be used to treat pulmonary disorders including acute lung injury (ALI); Acute respiratory distress syndrome (ARDS); Chronic obstructive pulmonary disease (COPD); And idiopathic pulmonary fibrosis (IPF). Rejecting antibodies to SDC2 for sepsis and sepsis-induced multiple organ failure, bone marrow transplant (BMT) or hematopoietic stem (HSC) rejection; Solid organ transplantation (SOT) rejection (including liver, kidney, skin, cornea, heart, lungs); Acute toxin-induced liver failure; Autoimmune hepatitis; Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC); Osteonecrosis; Degenerative disc disease; Rheumatoid arthritis; Osteoarthritis and delayed bone healing in diabetic patients; Autoimmune nephritis including Wegener's granulomatosis (WG); Burns, severe burns; Atrophic syndromes including muscle wasting disease and hypoxia; Other muscle wasting diseases including cachexia and muscular dystrophy (Duchenne and Becker); Congestive heart failure, acute myocardial infarction and stroke; Type 1 diabetes; Type 2 diabetes; Diabetic retinopathy and other retinopathies; Diabetic nephropathy and other nephropathies; Diabetic neuropathy and other neuropathies; Non-healing diabetic ulcer; Diabetic cardiomyopathy and other myopathies; Atherosclerosis; Peripheral arterial disease and severe ischemia; Uveitis; (Wet or dry) acute macular degeneration (AMD); Retinal and corneal damage; Autoimmune diseases such as autoimmune gastritis (AIG); Graft versus host disease (GvHD); Multiple sclerosis and dehydratative diseases; Thyroid disease; Inflammatory bowel disease including Crohn's disease, ulcerative colitis, and fistula Crohn's disease; Skin sclerosis; Lupus (SLE); Graves' disease; And autoimmune lymphoproliferative disorders (ALPS).

Antibodies against SDC2 can also be used for the treatment of a variety of endocrine disorders, such as dysmenorrhoeae, tendon injury and exercise-induced pulmonary hemorrhage (EIPH) (also known as "bleeding" or "bleeding episodes").

Antibodies useful in the present invention include antibodies that bind to SDC2 and also bind to a secondary target. Thus, these antibodies may comprise another antigen, e. G., A secondary binding domain that binds to a cell surface antigen, and may include a bispecific antibody as is generally known in the art.

The term "antibody" includes derivatives or functional fragments thereof that still retain binding specificity. Techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, 1999. The term "antibody" also includes immunoglobulins (Ig) of different classes (e.g., IgA, IgG, IgM, IgD and IgE) and subclasses (e.g., IgG1, IgG2, etc.).

The term "antibody" also encompasses embodiments such as antibody fragments, in particular Fab fragments, as well as chimeric, short chain and humanized antibodies. An antibody fragment or derivative can also be a VH or VL antibody that specifically binds to an antigen or epitope independently of an F (ab ') 2, Fv, scFv fragment or single domain antibody, a single variable domain antibody, Lt; RTI ID = 0.0 > variable domains. ≪ / RTI > Such immunoglobulin single variable domains include isolated polypeptide monoclonal variable domain polypeptides as well as larger polypeptides comprising one or more monomers of the antibody single variable domain polypeptide sequence.

References to human SDC2

(a) an amino acid sequence as set forth in SEQ ID NO: 3 or 4;

(b) natural variants of (a);

(c) a heterophasic body of (a) or (b)

(d) biologically active, diagnostic or therapeutically useful fragments, analogs, variants and derivatives thereof,

(e) the extracellular domain of (a) to (d), and

(f) all of the above dimers and oligomers

Or a polypeptide comprising the same.

The SDC2 polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably natural or recombinant polypeptides. The terms "fragment", "derivative", "variant" and "analog" refer to those which retain essentially the same biological function or activity as SDC2 and which retain (i) one or more of the amino acid residues Or (ii) one or more of the amino acid residues comprises a substituent, or (iii) the mature polypeptide is replaced by another compound, such as, but not limited to, the polypeptide (Iv) an additional amino acid may be fused to the mature polypeptide to facilitate purification of the mature polypeptide, for example, a compound which increases half-life (e. G., Polyethylene glycol or polysialic acid) ≪ / RTI >

The polypeptides of the present invention may have at least 60% similarity (preferably at least 60% similarity) with the polypeptides of SEQ ID NOS: 3 and 4 as well as the polypeptides of SEQ ID NOS: 3 or 4, At least 90% similarity (more preferably at least 80% identity) with the polypeptide of SEQ ID NO: 3 or 4, and more preferably at least 90% identity (more preferably at least 90% identity) Preferably further comprising a polypeptide having at least 95% similarity (even more preferably at least 95% similarity) with the polypeptide of SEQ ID NO: 3 or 4, and which generally comprises at least 100 amino acids Quot; includes at least 120 and more preferably at least 140 amino acids of such a portion of the polypeptide. The "similarity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitution to the sequence of the second polypeptide. A variety of different approaches are known for the calculation of sequence similarity and correspondence. Generally, a suitable way to perform this calculation is to use a program such as Smith-Waterman, BLAST, or FASTA to perform a database search and use one or preferably two or even three similarity tables . Blosum and PAM (point tolerated mutation) matrices are suitable amino acid similarity matrices for database search and sequence alignment. When using Smith-Waterman or FASTA it is appropriate to ensure that the break points are wide enough and if the initial run can not reveal any homology sequences it may be appropriate to try different operations, This is especially true if you start with either BLAST or FASTA.

In a particular embodiment of the present invention described in more detail in the following examples, a fragment of SDC2 was prepared and tested for its inherent, i.e., complete, activity against human SDC2. Active fragments were identified. Thus, the present invention also provides fragments of SDC2 that retain SDC2 activity, wherein said fragments are useful in the assay of Example 2 or in the assay of Example 9 (i.e., producing a dose-dependent inhibition of NFkB activation by TNF [ Quot; will exemplify the characteristic activity (although not necessarily the same efficacy) of human SDC2 in the context of the present invention. The active fragment retains SDC2 activity and can exceed the activity of native SDC2; The fragment preferably retains at least 30%, at least 50%, at least 70%, or at least 80% of the activity of native SDC2.

Accordingly, the present invention provides a polypeptide comprising or consisting of a fragment of SDC2, wherein said polypeptide has SDC2 activity. Said fragment suitably comprises no more than 150, no more than 120, no more than 100 or no more than 80 amino acids of SEQ ID NO: 3. In addition, the fragment of the present invention may separately comprise the signal sequence of SDC2, i.e. amino acids 1 to 18 of SEQ ID NO: 3. The fragment is suitably at least 30, at least 40, at least 50, or at least 60 amino acids in length. The use of said fragment, the composition comprising said fragment, and the method of treatment using said fragment are as for complete SDC2. Said fragment constitutes an embodiment of SDC2.

Preferably, the term SDC2 refers to a native ("complete") SDC2-type surface protein recognized by an active fragment of SDC2, an active variant of SDC2 and an antibody to SDC2, preferably an SDC2-specific antibody; Preferably, reference to SDC2 refers to native SDC2 and its active fragment, more preferably native SDC2. SDC2 antibody is found as catalog no. MAB29651 (clone 305507), available from R & D Systems, Inc., which is reactive with human, mouse, rat, horse, rabbit and swine. Human SDC2 according to the present invention is tested positively in the assay for immunosuppression according to Example 2 or 9 of the present invention, and more preferably binds to the SDC2 antibody.

Still further, the present invention provides an antibody to a polypeptide comprising or consisting of a fragment of SDC2. Such antibodies are suitable for use in human therapy, particularly for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing or (v) . Such antibodies are also suitable for therapies as disclosed herein for antibodies to the full SCD2.

order

SEQ ID NO: 1 - SDC2  human mRNA

SEQ ID NO: 2 - SEQ ID NO: 1 Nucleotide  605-1210

(Encodes SEQ ID NO: 3)

SEQ ID NO: 3 - SDC2  Human protein

SEQ ID NO: 4 - amino acids 19-201 of SEQ ID NO: 3

(Mature protein)

The invention is now described in the following embodiments, illustrated by the accompanying drawings, in which:
Figure 1 shows the detection of diverted SDC2 in the supernatant of Ad.SDC2 expressing MSC using ELISA;
Figure 2 shows that overexpression of Ad.SDC2 results in overexpression of SDC2 protein that increases over 72 hours;
Figure 3 shows that the Ad.SDC2 MSC ("S2") has a significantly increased immunosuppressive activity compared to the parent MSC;
Figure 4 shows the analysis of cell culture supernatant containing SDC2 ("S2") for effect on T cell proliferation;
Figure 5 shows that the SDC2 protein inhibits NFkB activation by TNF [alpha] and IL1 [beta];
Figure 6 shows NFkB induction in response to activation by recombinant SDC2 and TNF [alpha] and IL1 [beta];
Figure 7 shows NFkB induction in response to overexpression of SDC2 upon activation by TNF [alpha] and IL1 [beta];
Figure 8 shows that the nutrient-3a treatment of MSC results in a dose-dependent increase in SDC2 divergence;
Figure 9 shows that chemotherapy augments SDC2 divergence from human MSCs;
Figure 10 shows overexpression of the SDC-2 C-terminal deletion fragment (1-6) attenuates NF-kB activity in response to IL-1 [beta] or TNF- [alpha];
Figure 11 shows that adenoviral expression of SDC-2 attenuates NF-kB activity and IL6 / IL8 secretion in response to IL-1 [beta], TNF [alpha] and IL-1 [beta] / TNFa.

Example  1 - Increased SDC2  Immunosuppression by expression

Way

Transduction of MSC

MSCs were plated at a density of 10 ⁵ cells per well of a 6-well plate (Nunc) in complete medium (? -MEM, 10% FBS) and allowed to attach overnight. The cells were either leave as a negative control did not transduced or transduced with 1 ㎕ adenovirus (AD5 series) (10 ¹² vp / ㎖) encoding the human SDC2 rotated at 800 xg for 90 minutes, the plates at 37 ℃. The virus was placed on the cells for approximately 4 hours and then washed and replaced with serum-free medium (1 ml / well).

Western blot analysis

Cells were harvested by trypsinization and lysed in cell lysis buffer containing 50 mM Tris pH 7.4, 10% glycerol, 0.5% NP40, 150 mM NaCl and complete miniprotease inhibitor (Roche). Cell lysates were subjected to 10% SDS-PAGE; 50 μg protein per track was loaded. Protein detection was performed by 1: 500 dilution of anti-human syndecan-2 Ab (R & D Systems) in TBS 0.1% Tween, 3% BSA. A secondary anti-rat IgG antibody conjugated to horseradish peroxidase (Santa Cruz) was added at a dilution ratio of 1: 1000. Detection was then performed using ECL Western Blot chemiluminescence reagent (Pierce) and Flourochem imaging system.

Enzyme-Linked Immunosorbent Assay

Levels of human sindecane-2 were measured in supernatants collected from hMSCs using enzyme-linked immunoabsorption assays. The level of SDC2 was measured using a commercially available ELISA assay (CUSABIO). The analysis was performed according to the manufacturer's instructions. Calibration curves were generated using a refined standard for SDC2. Curve matching was performed by s-logistic regression analysis according to the manufacturer's instructions.

Isolation of human peripheral blood mononuclear cells

To isolate peripheral blood mononuclear cells (PBMCs), the anticoagulated blood samples were collected (7 to 8 mL), laminated on liquid density gradient media (GE Healthcare), centrifuged at 400 xg for 30 minutes at room temperature Separated. The upper layer was aspirated and discarded and PBMCs were harvested by careful pipetting of the corresponding dense contact interface layer (Buffy Coat) and transferred to a fresh 50 ml tube. The PBMCs were washed twice with 20 ml of PBS and centrifuged at 400 x g for 10 minutes. The platelets were then removed by slow centrifugation once at 200 x g for 10 minutes. The PBMCs were cultured in a T-cell culture medium (RPMI-1640, Gibco) containing 10% FBS, 50 μM β-mercaptoethanol, 1% NEAA and 1% L-glutamine in a Roswell Park Memorial Institute (RPMI) Lt; / RTI > The suspension of 10 ㎕ aliquots was recovered and the cell counts were determined using a hemocytometer.

Human T-cell proliferation assay

Human PBMC were washed with 0.1% BSA / PBS and incubated with 10 μM Vybrant carboxyfluorescein diacetate, succinimidyl ester (CFSE) / PBS staining solution (Invitrogen), pre-warmed (37 ° C.) At a concentration of 2 x 10 < ⁷ > cells / ml. The cells were incubated at 37 ° C for 6 minutes with blocking of light, and the reaction was stopped by adding 5 volumes of ice-cold medium containing 10% FBS. The PBMCs were washed three times with the culture medium to remove any remaining unbound CFSE. 100,000 CFSE stained PBMCs were stimulated with anti-human CD3 / anti-human CD28 soluble polyclonal antibodies in T-cell medium in 96-well round bottom plates. Various ratios of MSC were then added to the stimulated PBMC (1:10, 1:50, 1: 100, 1: 200 and 1: 400). Unstimulated PBMC were also cultured as a control. After 4 days, the PBMCs were harvested and then the supernatant was removed and the cells were washed in 100 [mu] l of autoclave washing solution containing 2% FBS. Subsequently, contrast dyeing with anti-human CD4 + -APC was performed. CFSE fluorescence of PBMCs was analyzed using FACSCanto. All proliferation was analyzed and compared to stimulated PBMC in the absence of MSC co-culture.

result

Name . SDC2  Overexpression MSC  Emanate from the supernatant SDC2 SDC2  By ELISA Detectable

At 24 hours after transduction with Ad.EGFP or Ad.SDC2, the medium was aspirated and replaced with serum-free medium and collected 24, 48 and 72 hours later. The supernatant was used as a control in an SDC2 ELISA with fresh serum-free medium.

Sindecane is a process known as divergence, usually undergoing controlled cleavage near the plasma membrane. The extracellular domain release of the sindecane can not only down-regulate signal transduction but also convert membrane-bound receptors into soluble effectors and / or antagonists (Manon-Jensen et al., 2010).

Using the human specificity SDC2 ELISA technique, we were able to detect and quantitate the SDC2 protein expressed by huMSC. We measured levels of diverted SDC2 protein in the culture medium of MSC expressing both Ad.EGFP and Ad.SDC2. SDC2 was detected in the culture medium of Ad.EGFP cells (346.4 pg / ml) after 48 hours and increased to 689.2 pg / ml after 72 hours. The SDC2-overexpressed cells showed an elevated level of divergent SDC2 (456 pg / ml) at 48 hours in the culture medium, which was approximately twice that of Ad.EGFP at 72 hours (1412.7 pg / ml) 1).

Referring to Figure 1, at 24 hours after transfection, the complete medium was exchanged with serum free (1 ml / well). The serum-free supernatant was collected after 48 hours and 72 hours. A commercially available SDC2 ELISA kit was used to detect the diverted SDC2 present in the supernatant. The Ad.SDC2 supernatant showed an increased amount of released SDC2 (~ 2-fold) compared to Ad.EGFP expressing cells.

The SDC2 protein expression was then tested to ensure that serum-free medium did not affect overexpression.

Name . SDC2  Overexpression is high SDC2  Protein expression Gt;

HuMSC was seeded at a density of 1 x 10 ⁵ cells / well in a 6-well plate and allowed to attach for 24 hours. HuMSC was then transfected with Ad.EGFP or Ad.S2 (1 x 10 ¹² vp / ml). At 24 hours after transfection, complete medium was replaced with serum-free medium. Protein lysates were harvested 24, 48, and 72 hours later. Western blotting (R & D) on SDC2 was performed on these lysates and found to significantly increase SDC2 protein expression. We observed an increase in the SDC2 band corresponding to the molecular weight of the core protein (~25 kDa) and the dimerized form (~ 48 kDa) (see Figure 2).

Immunosuppressive potential Of SDC2  Overexpression Increased

After confirming that SDC2 protein expression was increased by Ad.SDC2 transduction and also the soluble form of SDC2 emanated from the cell surface, we found that SDC2 overexpression may have an impact on the immunosuppressive potential of huMSC (if present ) Were investigated.

The immunosuppressive potential of the parent Ad.EGFP and Ad.SDC2 hMSC cells was evaluated using T-cell proliferation assay. The proliferation of the T-cell (CD4 +) fraction of PBMCs was measured by in situ cell assays of CFSE expression. CFSE is a fluorescent cell staining dye that is used to evaluate cell proliferation by progressively bisecting the cell's fluorescence with daughter cells following each cell division. Immunosuppression of the stimulated T-cells due to the presence of MSC causes inhibition of proliferation. Which was significantly reduced in the presence of Ad.SDC2 overexpressed MSC. The results are expressed as the percentage of proliferation over three generations. At a 1: 200 MSC: PBMC ratio, Ad.SDC2 MSC showed a significant T-cell immunosuppressive potential compared to the stimulated T-cell positive control (similar results were observed at 1:10, 1:50 and 1: 100 Rain). Ad.EGFP cells showed comparable levels of T-cell immunosuppression compared to parent MSC (see FIG. 3).

3, the immunosuppressive potential of the parent Ad.EGFP and Ad.SDC2 cell populations was assessed by co-culture with stimulated T cells and quantitated by stromal cell count. The results showed significant immunosuppression by Ad.SDC2 cells, which could inhibit T-cell proliferation compared to the stimulated T-cell positive control. Although the Ad.EGFP population retained equivalent immunosuppressive potential for parent MSC, the Ad.SDC2 population (denoted "S2 ") exhibited a significantly increased immunosuppressive potential for both parental and Ad.EGFP MSCs (* = P? 0.05, ** = P? 0.001 when measured using an independent sample T test).

Thus, overexpression of SDC2 in huMSC produced an enhanced immunosuppressive effect compared to huMSC.

Example  2 - SDC2  Overexpressing cells ( MSC ) From  Supernatant inhibited T cell proliferation

We analyzed the supernatant from a population of MSC overexpressing SDC2 for immunosuppressive activity.

4, the results show that the medium from the cells of the present invention, S2-overexpressing MSC ("S2") was stimulated with two control groups: (1) parent MSC ("parent") and (2) CD3 / CD28 CD28 < / RTI > induced T-cell proliferation compared to the medium from T cells ("T cell St"). Thus, the supernatant was immunosuppressive.

Example 3

As an efficacy analysis for therapeutic products SDC2  Analysis protocol

We developed a protocol for product efficacy testing based on SDC2 analysis.

The protocol

1. Analyze SDC2 levels among potential products

2. Compare with the pre-measured minimum values for acceptable therapeutic products

a. Level above the minimum value = allowed (end of test)

b. Below this minimum level = not allowed

3. Compare the maximum value for an unacceptable therapeutic product

a. Below this maximum level = rejected (end of test)

b. The median level between the minimum for an acceptable therapeutic product and the maximum for an unacceptable therapeutic product is increased to increase the SDC2 level and then the assay is repeated

Example 4

As an efficacy analysis for therapeutic products SDC2  analysis

We performed the protocol of Example 3 with an analysis specific to human cell preparations.

The protocol was used at the following pre-determined levels:

1. We analyzed SDC2 levels in potential products

2. An agent with a SDC2 level of greater than 1400 pg / ml was allowed to be suitable for further processing as a potential therapeutic product; No formulation below this level was allowed

3. Formulations with SDC2 levels of less than 500 pg / ml were initially recorded as not suitable for further processing

4. Formulations revealed to be intermediate between 500 and 1400 pg / ml were shown to be suitable for treatment with SDC2 actives to increase their SDC2 levels

5. Subsequently, we also noted that preparations with SDC2 levels of even less than 500 pg / ml could be treated with SDC2 activators to increase their SDC2 levels

6. The treated population with an elevated level of SDC2 above 1400 pg / ml was then allowed to be suitable for further processing as a potential therapeutic product.

Example  5 - Endogenous SDC2  Activation of p53 to increase

We tested various HDAC inhibitors for their effects on SDC2 expression on human cell populations initially isolated and subsequently maintained in culture based on SDC2 expression.

Splitomycin, valproic acid, and 2-pyrrolidinone-n-butyric acid (PBA) all increased SDC2 RNA levels at 24 hours and were most strongly stimulated by splittomicin.

We have similarly tested the environmental toxin benzo [a] pyrene-7,8-diol-9,10-epoxide (BPDE) and found that 600 nM BPDE increased the SDC2 secretion approximately 3-fold.

Example  In the 6-donor cell population SDC2  level

Three human MSC donors (donor cell preparations) were tested for their SDC2 secretion. The results show that the two donors identified as good performers (labeled donors 109 and 110) had a high SDC2 while the third donor identified as a poor performer (donor 111) had a low SDC2 level, SDC2 analysis failed.

Example 7

In diabetic wound healing SDC2 +, SDC2 - and PA - SSC's  Comparison of topical administration

We compared the activity of various stem cell populations in wound healing models using Excellagen (a highly purified, formulated homogenate of fibrous small intestinal type I collagen).

In vivo experimental model

Male New Zealand White rabbits (3 to 3.5 kg) were used in the study. Diabetes was induced in the rabbit by administration of alloxan (150 mg / kg) through the feces of the ear. Serum glucose was tested daily using the Accucheck advantage strip (Roche). After 5 weeks of hyperglycemia, rabbits were anesthetized with xylazine and ketamine. A sterile, disposable 6-mm punch biopsy was used to create five wounds (three wounds on one ear and two wounds on the other ear). Each wound was treated with one of five random treatment groups:

1. No processing

2. Excelase (R) scaffold alone (25 l)

3. Excelase (R) (having a concentration of 25 [mu] l from the cell pellet resuspended in ExcelLane (R)) with a 1 x 10 ⁶ wild-type MSC,

4. EXCELASE® (25 μl solution from resuspended cell pellets in ExcelLane®) with a 1 x 10 ⁶ SDC2 positive ("SDC2 +") type MSC,

5. EXCELASE (TM) (25 μL solution from cell pellet resuspended in ExcelLane (R)) with 1 x 10 ⁶ SDC2 negative ("SDC2-") type MSC.

After application of the treatments, the wounds were covered with a polyurethane dressing agent (OpSite; Smith & Nephew) and the ears were closed and applied with an adhesive dressing agent (Operfix; ≪ / RTI > Medicare (Promedicare, Ireland). The study treatment was performed randomly for non-biased evaluation. Seven days later, rabbits were sacrificed with intravenous sodium pentobarbital (2 mL).

Results analysis

After one week of treatment, there was a significant difference in wound healing between the different treatment groups. Each wound was traced on the day of killing. A new wound was made on the day of killing and the percent reduction in wound area was calculated over one week.

Evaluation of wound closure percentage

On the day of killing, each wound was tracked six times. The area of each phase was measured using Cell B software (Olympus) and the average area was calculated. The percent reduction in wound area over one week in each treatment group was calculated.

Percentage of wound closure

A wound-shear percentage analysis found that the accelerator (s) accelerated wound healing compared to untreated wounds. Wounds treated with one million SDC2 + cells in the excelasin (R) scaffold exhibited the most significant wound closure percentage at best than the untreated group at week 1. The wound closure effect of EXCELAGEN (TM) was significantly enhanced when mixed with SDC2 + cells.

All wound sections were further treated for histological and serological analysis.

histology

The wound was cut across the midline and fixed in 10% formalin for 24 hours. The tissue was processed using an organism processor (ASP300; Meyer Instruments, Houston, Tex., USA) and embedded in paraffin. The compartment (5 mm) was stained with hematoxylin and eosin and methionine trichloromethane using standard protocols.

From the histological staining, it was observed that the wound treated with Excelsan (TM) showed good wound healing with the formation of new tissue on wounded area. Wounds treated with SDC2 + cells mixed with Excelsan (TM) showed the most effective wound healing. The mixing of SDC2 + cells increased the healing potential of excelagen (TM) alone. Compared to the untreated group, SDC2 + treated wounds showed collagen formation at the base of wound layer and formation of new tissue on scarred sites.

Angiogenesis in wound beds with SDC2 + cell treated wounds

MSCs are known to promote angiogenesis in addition to improving skin wound healing. Similar effects were observed in wound treated with SDC2 + cells. In the wound, formation of new blood vessels could be observed within the wound layer. Thus, prominent wound healing was observed by an increased wound closure percentage in SDC2 + treated wounds, which may be associated with more efficient angiogenesis.

summary

MSC mixed with EXCELASE (TM) maintained good metabolic activity, reflecting no side effects of the EXCELASE (TM) substrate on cell viability. The cells were densely present throughout the excelasal (TM) substrate without any cell mass formation with proper distribution. In current studies, scars treated with SDC2 + cells mixed with Excelsan (TM) exhibited an increased wound closure percentage compared to the excellance (TM) treated control alone. The SDC2 + cells markedly increased the wound healing potential of Xcellan (TM). MSCs are also reported to promote angiogenesis. In this study, increased angiogenesis was observed within the wound layer in the SDC2 + cell treatment group. Thus, SDC2 + treated wounds exhibited significant wound healing benefits due to increased wound closure percentage with more prominent angiogenesis. Thus, SDC2 + cells of the present invention in the substrate exhibited improved wound healing potential with less healing time.

Example  8 - Cancer cell migration

Cell migration is an important parameter for in vitro cell culture-related studies. It is necessary to monitor the dynamic changes of the cell population under different conditions. Using a real-time cell analyzer (RTCA, xCELLigence, Roche), an impedance-based device, we can monitor real-time the following properties: proliferation, migration and cell attachment. Such properties relate to cancer development. We have established the ability of the cancer cells to migrate towards the conditioned medium using the above method. The model is allowed as a prelude to efficacy in cancer therapy, i.e. a reduced migration in the model indicates anticancer properties.

In our study, cell migration was measured using real-time 16-well CIM plate readings using the xCELLigence device mentioned above. A variety of cancer cell lines from breast cancer (MDA-MB-231, MDA-MB-486, MCF-10A), prostate cancer (DU145), pancreatic cancer (SU-86-86) Respectively.

The results are summarized in Table 1:

Cancer cell migration
Cell type Amount of recombinant SDC2 100 ng / ml 500 ng / ml 1000 ng / ml Ad.SDC2 (3 [mu] L) MDA-MB-231 ↓ ↓ ↓ ↓ ↓ ↓ ↓ - ↓↓↓ MDA-MB-486 ↓ MCF-10A - Du145 ↓ - ↓ SU-86-86 ↓ ↑ ↓ ↓ ↓ ↑ ↓ HCT116 ↓ ↓ ↓ Note) ↑ = increased movement compared to conditioned medium;
↓ = reduced cell migration compared to conditioned medium;
- = No change compared to conditioned medium

Breast cancer cell line

Recombinant SDC2 (500 ng / ml rSDC2 (n = 5) and 100 ng / ml (n = 2) inhibited the migration of MDA-MB-231 cells. Similar migration results were generated by Ad.SDC2 (3 [mu] l) -CM (n = 3).

MDA-MB-486 cells showed a decrease in migration towards Ad.SDC2-CM (3 [mu] l; n = 1).

Ad.SDC2 (3 [mu] L) did not alter the migration of MCF-10A cells (n = 1).

Prostate cancer cell line

Addition of 500 ng / ml of rSDC2 to serum-free MSC-CM reduced the migration of DU145 cells in one experiment compared to conditioned media alone, and no change in the second experiment. Ad.SDC2 (3 [mu] l) reduced the migration of DU145 cells (n = 1). Indicating that the EC domain of SDC2 inhibited the migration of the prostate cancer cell line.

Pancreatic cancer cell line

The migration capacity of SU-86-86 cells varied; One experiment increased migration towards rSDC2-CM (100 ng / ml, 500 ng / ml and 1000 ng / ml; n = 1) And 1000 ng / ml; n = 1).

colon carcinoma  Cell line

HCT116 cells showed a decrease in migration towards rSDC2-CM at all concentrations tested (100-1000 ng / ml).

conclusion

Collectively, these results indicate behavioral changes of different cancer cells towards SDC2 in the medium. In general, SDC2 reduced the ability of the cancer to migrate, indicating the anticancer effect in this model, particularly MDA-MB-231 cells (breast cancer).

In the initial study, subsequent antibodies to SDC2, as evidenced by an expanded research program in the field, showed a similar inhibition of breast cancer migration, indicating anti-cancer activity.

Example  9 - immunosuppression

Using rSDC2 as used in Example 8, we investigated the activity of SDC2 in a model of immunosuppression. We found that SDC2 protein inhibits NFkB activation by TNFα and IL1β. This demonstrated the immunosuppressive activity of the SDC2 protein itself.

A549 cells stably transfected with the kB luciferase plasmid were pre-treated with SDC2 recombinant protein for 1 hour. 10 ng / ml of TNF-α / IL-1β was added to the medium and luciferase assay was performed after 24 hours. The results are shown in Fig. *** = 0 ng / ml p < 0.0001 for S2.

A549 cells were stably transfected with the kB luciferase plasmid and treated with recombinant SDC2 for 24 hours. The results are shown in Fig. In the upper panel: Cytokine was added to the medium for 24 hours and luciferase assay was performed. In the donor panel: After replacing the medium, cytokines were added for 24 hours. * = 0 ng / ml p < 0.01 for S2; *** = 0 ng / ml p < 0.0001 for S2; +++ = 100 ng / ml p < 0.0001 for S2.

Luciferase assay was performed after transfection of A549 kBL cells with 1 [mu] l / ml or 3 [mu] l / ml Ad.Null or Ad.SDC2 and treatment with cytokine for 24 hours. The results are shown in Fig. ** = 0 ng / ml p < 0.001 for S2.

The results showed immunosuppressive effect of SDC2 protein.

Example  10 - Of SDC2  p53 regulation

To determine the role of p53 in SDC2 protein signaling, we pharmacologically disturbed the p53 pathway in the MSC using the p53 agonist (nutriin-3a) to aid in the detailed description of the SDC2 response. Nutuline-3a induces a significant p53 response and inhibits growth.

Way

Cell culture and treatment

MSCs were plated at a density of 10 ⁵ cells per well of a 6-well plate (nank) in complete medium (a-MEM, 10% FBS) and allowed to attach overnight. The medium was then exchanged with serum-free nuululine-3a or DMSO as a carrier control. At 24 hours after introduction, the cells were harvested for both RNA and protein and the serum-free supernatant was collected for ELISA.

Enzyme-Linked Immunosorbent Assay (ELISA)

Levels of human sindecane-2 were measured in supernatants collected from hMSC using commercially available ELISA assay (CUSABIO). The analysis was performed according to the manufacturer's instructions. Calibration curves were generated using a refined standard for SDC2. Curve matching was performed by s-logistic regression analysis according to the manufacturer's instructions.

result

MSC Nutlin -3a treatment SDC2  A dose-dependent increase in divergence Generate

24 hours after treatment with Nutriin-3a (5, 10, 20 [mu] M) or DMSO carrier control (5, 20 [mu] M), the supernatant from MSC was collected, centrifuged for 5 minutes at 1500 rpm, SDC2 &lt; / RTI &gt; ELISA was performed with unconditioned serum-free medium.

Figure 8 shows that nutrien-3a treatment of MSC results in a dose-dependent increase in SDC2 divergence. MSCs were treated with neutrin-3a or DMSO as a carrier control for 24 h in serum-free medium. The serum-free supernatant was collected and SDC2 ELISA kit available commercially was used to detect the diverted SDC2 present in the supernatant. Cells treated with the nutrien-3a showed increased SDC2 divergence compared to the DMSO control.

Figure 8 shows three independent experiments for three different human MSC donors. The amount of SDC2 released is increased from 250 pg / ml in the 20 μM DMSO control to almost 1000 pg / ml in the 20 μM dose of nutriin-3a.

Thus, this experiment demonstrates that the compound that upregulates p53 activity (nullrin-3a) also increases SDC2 divergence.

Example 11

The chemotherapeutic agent is human From the MSC SDC2  Divergence Increase

We have investigated the effects of SDC2 divergence from human MSC on various chemotherapy inhibitors; In particular, Taxol, camptothecin, etoposide and BPDE (benzo (a) pyrenediol epoxide) were tested.

The MSCs were treated with the chemotherapeutic agents for 24 hours before serum starvation for 24 hours. The cell supernatant was harvested and the relative change in folds of SDC2 protein expression was analyzed by ELISA.

Figure 9 shows the results of this experiment and shows that chemotherapeutics increase SDC2 divergence from human MSCs.

Example 12

NF - of kB  Coordinating SDC Domain verification for -2

To determine that fragments or domains of SDC-2 may be responsible for the regulation of NF-kB signaling, we used the luciferase reporter gene system under the control of kB motility. We transduced the cells with an empty vector-mediated adenovirus (p3xFLAG-CMV-14) or a vector expressing 12 fragments SDC2 (1.4-12.4) and measured the effect on NF-kB transcriptional activity.

Figure 10 shows that overexpression of SDC-2 C-terminal deletion fragments (1 to 6) attenuates NF-kB activity in response to IL-1 [beta] or TNF- [alpha]. NF-kB-luciferase-expressing cells were transfected with a vector expressing the empty vector (p3xFLAG-CMV-14) or fragments 1.4 to 12.4. After 24 hours, cells were treated with cytokines, IL-1 beta (10 ng / ml), TNF-alpha (10 ng / ml) for 24 hours and then luciferase assay was performed.

These fragments are referred to as numbers 1 through 12 in FIG. These correspond to the following peptide fragments of SDC-2: 1,1-79; 2,1-87; 3.1-100; 4.1-144; 5.1-169; 6.1-201; 7,19-79; 8,19-87; 9, 19-100; 10, 19-144; 11, 19-169; 12,19-201

Pre-inflammatory cytokines such as TNF-a and IL-l [beta] can activate NF-kB transcriptional activity; This is observed in the empty vector control lane. NF-kB transcriptional activity is approximately 3- and 4-fold inducible by TNF- [alpha] and IL-1 [beta] in control cells, respectively. However, the TNF-a-, IL-l [beta] and TNF-alpha / IL-l [beta] -induced increases of NF-kB transcriptional activity in fragments 1-6 expressing cells are markedly reduced (Fig. 10). Significantly, the fragments expressing the N-terminal signal peptide (1-18) significantly inhibited TNF-α and IL-1β activity of NF-kB. Fragments 1.4 (1-79) and 2.4 (1-87) firmly suppressed NF-kB activation suggesting that both heparan sulfate binding site and signal peptide are required for inhibition of NFkB.

The data suggest that the immunosuppressive properties of SDC2, and possibly the migration properties, could be due to the N-terminal of SDC-2. By inhibiting NF-kB activity, SDC2 fragments can reduce the immune response and can be used as anti-inflammatory agents, and in wound healing, cancer therapy and inflammatory diseases.

Example 13

Sindecan -2 is TNF -α, IL - 1β  And TNF -α / IL - 1β  Reactive NF - kB  Inhibit signal transduction and release IL6 and IL8 secretion Limit

To determine that SDC-2 could affect NF-kB signaling, we used the luciferase reporter gene system under the control of kB motif. We transduced the cells with an empty vector-mediated adenovirus (Ad-control) or SDC2 expressing adenovirus (Ad-SDC2) and measured the effect on NF-kB transcriptional activity. Pre-inflammatory cytokines, such as a combination of TNF- [alpha], IL-1 [beta] and TNF- [alpha] / IL-1 [beta] can activate NF- [kappa] B transcriptional activity as shown in FIG. 11A. NF-kB transcriptional activity is approximately 3-, 3.5- and 4-fold inducible by TNF- [alpha], IL-l [beta] and TNF-alpha / IL-l [beta] in control cells, respectively. However, the TNF- [alpha], IL-l [beta] and TNF-alpha / IL-l [beta] -induced increase of NF-kB transcriptional activity in SDC-2 expressing cells is markedly reduced (Fig. 11).

In addition to the reduction of NF-kB transcriptional activity, TNF-α-, IL-1β- and TNF-α / IL-1β-induced release of IL-6 and IL-8 (both of which are regulated by NF-kB) There was a significant reduction associated (Figs. 11B and 11C).

Taken together, this data indicates the anti-inflammatory properties of SDC2 by modulation of the NF-kB pathway.

Example 14

Targeting SDC2 Antibodies to Localized SDC2 in Cancer Substrate

We investigated the localization of SDC2 ⁺ cells in the cancer model to identify earlier studies in cancer treatment and to determine strategies for therapeutic intervention.

The MMTV PyMT-P2A-mCherry-P2A-OVA (PyMT ChOVA) mouse was kindly created by USCF professor Matt Krummel. Wherein the mCherry and OVA (ovalbumin) sequences are joined to a polyoma virus, a middle T antigen (PyMT) and the entire sequence is driven by the MMTV (mouse breast tumor virus) promoter.

Tumor growth-female PyMT ChOVA mice were monitored for tumor initiation by mammary gland activation and monitored weekly for total tumor size by measurement of tumor size by vernier calipers. The combined tumor size was calculated by weighting all promotable tumor areas. The tumor area was defined as the length x width of the tumor. Mice were killed according to the ACREC animal protocol when tumor size exceeded 200 mm &lt; 2 &gt;.

Tumor cleavage - PyMT tumors were excised from the mice and the total weight of the removed tumors was determined. The tumors were then fixed with a scalpel and cut with 2 mg / ml collagenase IV (Sigma) and 200 [mu] g / ml DNAse per 0.3 gram tumor weight for 1.5 hours. The tumor was then passed through a 100 탆 cell strainer to remove large uncut pieces of tumor. A 70% / 37% / 30% Percoll gradient was then performed to remove dead cells and red blood cells, and the two contact interfaces were collected.

Inoculated cell counts - All antibodies were purchased from R & D Systems, BD Pharmingen, eBioscience, Invitrogen or Biolegend. For surface staining, cells were incubated with anti-Fc receptor antibody (24G2) and stained with a suitable antibody in PBS 2% FCS. All stained cell counts were performed on a FACSCanto stromal cell line (BD Biosciences). Analysis of stained cell count data was performed using FlowJo (Treestar).

Markers of CD45 to a tumor cell substrate ^-, mCherry ^- were classified on the basis of ^-, gp38 ^+, CD362 ⁺ and DAPI. Epithelial tumor cells were classified based on CD45 ^- , DAPI ^-, and mCherry ⁺ .

The results SDC2 (CD362 / Shin-decane-2) protein expression in CD45 ^- show that, gp38 ^+, CD362 ^+, and DAPI to localize the tumor stroma ^-, mCherry. Thus, SDC2-positive cells were localized in the cancerous substrate, which further supports early studies suggesting the anti-cancer effects of antibodies to SDC2 antagonists, such as SDC2, in cancer therapy.

Thus, the present invention provides immunomodulation and other therapies through modulation of SDC2 levels and / or activity.

                         SEQUENCE LISTING <110> Orbsen Therapeutics Limited   <120> Immuno-modulatory and Therapeutic Agents and Compositions, and        Uses Thereof <130> P39937WO <150> GB 1306886.1 <151> 2013-04-16 <150> GB 1314544.6 <151> 2013-08-14 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 3491 <212> DNA <213> Homo sapiens <400> 1 gcccggagaa gcaggctcag gagggaggga gccagaggaa aagaagagga ggagaaggag 60 gaggacccgg ggagggaggc gcggcgcggg aggaggaggg gcgcagccgc ggagccagtg 120 gccccgcttg gacgcgctgc tctccagata cccccggagc tccagccgcg cggatcgcgc 180 gctcccgccg ctctgcccct aaacttctgc cgtagctccc tttcaagcca gcgaatttat 240 tccttaaaac cagaaactga acctcggcac gggaaaggag tccgcggagg agcaaaacca 300 cagcagagca agaagagctt cagagagcag ccttcccgga gcaccaactc cgtgtcggga 360 gtgcagaaac caacaagtga gagggcgccg cgttcccggg gcgcagctgc gggcggcggg 420 agcaggcgca ggaggaggaa gcgagcgccc ccgagccccg agcccgagtc cccgagcctg 480 agccgcaatc gctgcggtac tctgctccgg attcgtgtgc gcgggctgcg ccgagcgctg 540 ggcaggaggc ttcgttttgc cctggttgca agcagcggct gggagcagcc ggtccctggg 600 gaatatgcgg cgcgcgtgga tcctgctcac cttgggcttg gtggcctgcg tgtcggcgga 660 gtcgagagca gagctgacat ctgataaaga catgtacctt gacaacagct ccattgaaga 720 agcttcagga gtgtatccta ttgatgacga tgactacgct tctgcgtctg gctcgggagc 780 tgatgaggat gtagagagtc cagagctgac aacatctcga ccacttccaa agatactgtt 840 gactagtgct gctccaaaag tggaaaccac gacgctgaat atacagaaca agatacctgc 900 tcagacaaag tcacctgaag aaactgataa agagaaagtt cacctctctg actcagaaag 960 gaaaatggac ccagccgaag aggatacaaa tgtgtatact gagaaacact cagacagtct 1020 gtttaaacgg acagaagtcc tagcagctgt cattgctggt ggagttattg gctttctctt 1080 tgcaattttt cttatcctgc tgttggtgta tcgcatgaga aagaaggatg aaggaagcta 1140 tgaccttgga gaacgcaaac catccagtgc tgcttatcag aaggcaccta ctaaggagtt 1200 ttatgcgtaa aactccaact tagtgtctct atttatgaga tcactgaact tttcaaaata 1260 aagcttttgc atagaataat gaagatcttt gttttttgtt ttcattaaag agccattctg 1320 gcactttaat gataaaatcc cattgtattt aaaacatttc atgtatttct ttagaacaac 1380 ataaaattaa aatttaacat ctgcagtgtt ctgtgaatag cagtggcaaa atattatgtt 1440 atgaaaaccc tcgatgttca tggaattggt ttaaactttt atgcgcaaat acaaaatgat 1500 tgtctttttc ctatgactca aagatgaaag ctgtttcatt tgtgtcagca tgtctcagat 1560 tgaccttacc aagttggtct tactttgtta atttatctgt tgtccccttc ctctcctctg 1620 ccctcccttc ttgtgccctt aaaaccaaac cctatgcctt ttgtagctgt catggtgcaa 1680 tttgtctttg gaaaattcag ataatggtaa tttagtgtat atgtgatttt caaatatgta 1740 aactttaact tccactttgt ataaattttt aagtgtcaga ctatccattt tacacttgct 1800 ttatttttca ttacctgtag ctttgggcag atttgcaaca gcaaattaat gtgtaaaatt 1860 ggattattac tacaaaaccg tttagtcata tctatctaat cagatcttct tttgggagga 1920 tttgatgtaa gttactgaca agcctcagca aacccaaaga tgttaacagt attttaagaa 1980 gttgctgcag attcctttgg ccactgtatt tgttaatttc ttgcaatttg aaggtacgag 2040 tagaggttta aagaaaaatc agtttttgtt cttaaaaatg catttaagtt gtaaacgtct 2100 ttttaagcct ttgaagtgcc tctgattcta tgtaacttgt tgcagactgg tgttaatgag 2160 tatatgtaac agtttaaaaa aaaagttggt attttataag cacagacaat tctaatggta 2220 acttttgtag tcttatgaat agacataaat tgtaatttgg gaacataaaa actactgaat 2280 aaatcatgtg gcctaatatt gaaaatgtca ctgttataaa ttttgtacat ttttgatcaa 2340 atgtacatct cccctttgct aacggccgtc tgctctcaag gatgacgtgg gtttgatttc 2400 taagtgtttc acagtgtctg taaatcaaga ccaaagagcc tgtcgatgag actgtttatt 2460 accagattca cttctgaatt ggccagagga aatctgaatg tattatcctg tgtgtgtcta 2520 ggtagagata ttggaaggct gccaggggat ttcgaagttt gcaaccttta taggataact 2580 gatggcaata ttaagacaga cgcctgcttt tgcaaataac ttacaagact gtaaattcca 2640 aagatctgaa tggggctttc ctgatgttgg tatctaaggc ttaggcctat agattgattt 2700 acctttggaa ttgtgctcca aatgtctact gaagcttaac cgaagaacta ataaatggac 2760 tacagtagct cacgttacag ggaaggaggg taggcaggga ggctctgtgt gttaaaatga 2820 gggtctcact gctttaggat tgaagtggct ggaaagagtg atgcctgggg aaggagatgg 2880 agttatgagg gtactgtggc tggtactttc tgtactaaac atttcctttt tctattttac 2940 cactaatttt gttttaaact gtgagccgtc caagtcagaa gaagacagca aaaaaagcaa 3000 cttttccaac atacaattta cttttaataa agtatgaata tttcattttg agaacattcc 3060 ctggaattgc cacataattc attaaaaaca tttttttaag caacacttgg aacagtgttt 3120 actttaaatc cttaatggcc ttaattaatt ctcagattcc tgccccatca cttacagaac 3180 caattcactt tagagtgact aaaaggaaac gatagcctag ctttctaaag ccacgctgtg 3240 tccctcaatt acagagggta ggaatgggta tacctctaac tgtgcaaagc agagtgaaat 3300 tcaattcata gaataacaac tgctgggaat atccgtgcca ggaaaagaaa aatttctggc 3360 aaatattttg tcactgctgt aaagcaaaat atttgtgaaa gtgccaaaat aaagtctgtc 3420 atgccaaaag taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480 aaaaaaaaaa a 3491 <210> 2 <211> 606 <212> DNA <213> Homo sapiens <400> 2 atgcggcgcg cgtggatcct gctcaccttg ggcttggtgg cctgcgtgtc ggcggagtcg 60 agagcagagc tgacatctga taaagacatg taccttgaca acagctccat tgaagaagct 120 tcaggagtgt atcctattga tgacgatgac tacgcttctg cgtctggctc gggagctgat 180 gaggatgtag agagtccaga gctgacaaca tctcgaccac ttccaaagat actgttgact 240 agtgctgctc caaaagtgga aaccacgacg ctgaatatac agaacaagat acctgctcag 300 acaaagtcac ctgaagaaac tgataaagag aaagttcacc tctctgactc agaaaggaaa 360 atggacccag ccgaagagga tacaaatgtg tatactgaga aacactcaga cagtctgttt 420 aaacggacag aagtcctagc agctgtcatt gctggtggag ttattggctt tctctttgca 480 atttttctta tcctgctgtt ggtgtatcgc atgagaaaga aggatgaagg aagctatgac 540 cttggagaac gcaaaccatc cagtgctgct tatcagaagg cacctactaa ggagttttat 600 gcgtaa 606 <210> 3 <211> 201 <212> PRT <213> Homo sapiens <400> 3 Met Arg Arg Ala Trp Ile Leu Leu Thr Leu Gly Leu Val Ala Cys Val 1 5 10 15 Ser Ala Glu Ser Arg Ala Glu Leu Thr Ser Asp Lys Asp Met Tyr Leu             20 25 30 Asp Asn Ser Ser Ile Glu Glu Ala Ser Gly Val Tyr Pro Ile Asp Asp         35 40 45 Asp Asp Tyr Ala Ser Ala Ser Gly Ser Gly Ala Asp Glu Asp Val Glu     50 55 60 Ser Pro Glu Leu Thr Thr Ser Arg Pro Leu Pro Lys Ile Leu Leu Thr 65 70 75 80 Ser Ala Ala Pro Lys Val Glu Thr Thr Thr Leu Asn Ile Gln Asn Lys                 85 90 95 Ile Pro Ala Gln Thr Lys Ser Pro Glu Glu Thr Asp Lys Glu Lys Val             100 105 110 His Leu Ser Asp Ser Glu Arg Lys Met Asp Pro Ala Glu Glu Asp Thr         115 120 125 Asn Val Tyr Thr Glu Lys His Ser Asp Ser Leu Phe Lys Arg Thr Glu     130 135 140 Val Leu Ala Ala Val Ale Gly Gly Val Ile Gly Phe Leu Phe Ala 145 150 155 160 Ile Phe Leu Leu Leu Leu Val Tyr Arg Met Arg Lys Lys Asp Glu                 165 170 175 Gly Ser Tyr Asp Leu Gly Glu Arg Lys Pro Ser Ser Ala Ala Tyr Gln             180 185 190 Lys Ala Pro Thr Lys Glu Phe Tyr Ala         195 200 <210> 4 <211> 183 <212> PRT <213> Homo sapiens <400> 4 Glu Ser Arg Ala Glu Leu Thr Ser Asp Lys Asp Met Tyr Leu Asp Asn 1 5 10 15 Ser Ser Ile Glu Glu Ala Ser Gly Val Tyr Pro Ile Asp Asp Asp Asp             20 25 30 Tyr Ala Ser Ala Ser Gly Ser Gly Ala Asp Glu Asp Val Glu Ser Pro         35 40 45 Glu Leu Thr Thr Ser Arg Pro Leu Pro Lys Ile Leu Leu Thr Ser Ala     50 55 60 Ala Pro Lys Val Glu Thr Thr Thr Leu Asn Ile Gln Asn Lys Ile Pro 65 70 75 80 Ala Gln Thr Lys Ser Pro Glu Glu Thr Asp Lys Glu Lys Val His Leu                 85 90 95 Ser Asp Ser Glu Arg Lys Met Asp Pro Ala Glu Glu Asp Thr Asn Val             100 105 110 Tyr Thr Glu Lys His Ser Asp Ser Leu Phe Lys Arg Thr Glu Val Leu         115 120 125 Ala Ala Val Ile Aly Gly Gly Val Ile Gly Phe Leu Phe Ala Ile Phe     130 135 140 Leu Ile Leu Leu Leu Val Tyr Arg Met Arg Lys Lys Asp Glu Gly Ser 145 150 155 160 Tyr Asp Leu Gly Glu Arg Lys Pro Ser Ser Ala Ala Tyr Gln Lys Ala                 165 170 175 Pro Thr Lys Glu Phe Tyr Ala             180

Claims (75)

A composition comprising SDC2 or SDC2 for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. The method according to claim 1,
SDC2 or composition comprising soluble SDC2. 3. The method according to claim 1 or 2,
SDC2 or composition wherein SDC2 is human SDC2. 4. The method according to any one of claims 1 to 3,
SDC2 or composition comprising cell culture medium. 5. The method of claim 4,
SDC2 or composition wherein SDC2 is obtained from cell culture supernatant. a vector encoding SDC2 for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. The method according to claim 6,
Vector encoding human SDC2. A compound that modulates the expression of SDC2 for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. 9. The method of claim 8,
Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 9. The method of claim 8,
0.0 &gt; SDC2. &Lt; / RTI &gt; 11. The method of claim 10,
lt; RTI ID = 0.0 &gt; p53. &lt; / RTI &gt; 11. The method of claim 10,
0.0 &gt; ERK, p38 &lt; / RTI &gt; or JNK SAPK kinase pathway. 11. The method of claim 10,
A compound that is selected from compounds that activate a hypoxia-inducible factor (HIF) -mediated pathway. 11. The method of claim 10,
TGF, BMP, Lefty, Nodal, and Activin pathways. 11. The method of claim 10,
Compounds selected from compounds which function NOTCH pathways, for example Jagged1, Jagged2, DLL1, DLL2, DLL3 and DII4. 11. The method of claim 10,
A compound that is selected from compounds that functionalize the Hedgehog signaling pathway. 11. The method of claim 10,
Lt; RTI ID = 0.0 &gt; WNT &lt; / RTI &gt; pathway. 11. The method of claim 10,
A compound that activates an &lt; RTI ID = 0.0 &gt; androgen &lt; / RTI &gt; receptor or an estrogen receptor pathway. 11. The method of claim 10,
Lt; RTI ID = 0.0 &gt; NFkB &lt; / RTI &gt; pathway. Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 21. The method of claim 20,
A method of treating a population of cells such that the cell is suitable for use in: (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. 22. The method according to claim 20 or 21,
Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 22. The method according to claim 20 or 21,
Comprising contacting the cell with SDC2, or a composition, vector or compound according to any one of claims 1 to 19. 24. The method according to any one of claims 20 to 23,
Wherein the cell is a human cell and SDC2 is human SDC2. 25. The method according to any one of claims 20 to 24,
Wherein the cell is a stem cell. 26. The method of claim 25,
Wherein the cell is a stromal stem cell. 26. The method of claim 25,
Wherein the cell is an MSC. 28. The method according to any one of claims 20-27,
And deriving offspring cells or tissues from the treated cells. 28. A cell or tissue obtained from the method of claim 28. Lt; RTI ID = 0.0 &gt; SDC2 &lt; / RTI &gt; by the cell or cells. 31. The method of claim 30,
A method of treating a population of cells such that the cell is suitable for use in: (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. 32. The method according to claim 30 or 31,
Lt; RTI ID = 0.0 &gt; SDC2 &lt; / RTI &gt; to modulate the immunosuppressive properties of the cell. 33. The method according to any one of claims 30 to 32,
Lt; RTI ID = 0.0 &gt; SDC2 &lt; / RTI &gt; to increase the immunosuppressive properties of the cell. 34. The method according to any one of claims 30 to 33,
Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 35. The method according to any one of claims 30 to 34,
RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 36. The method according to any one of claims 30 to 35,
The method comprising treating the cell with an increase in expression of SDC2, e.g., by increasing the expression of the cell's exogenous SDC2, or by exposure to environmental conditions. 37. The method according to any one of claims 30-36,
A method wherein the expression of SDC2 by cells is low or not prior to treatment. 37. The method according to any one of claims 30 to 37,
Wherein the expression of SDC2 by the cell is initially high but decreases over time and the treatment restores the previous SDC2 expression level. 39. The method of claim 30 or claim 38,
Lt; RTI ID = 0.0 &gt; SDC2 &lt; / RTI &gt; or fragments thereof. 40. The method according to any one of claims 30-39,
Wherein the cell is a human cell and SDC2 is human SDC2. 41. The method according to any one of claims 34 to 40,
Wherein the cell is a stem cell. 42. The method of claim 41,
Wherein the cell is a stromal stem cell. 42. The method of claim 41,
Wherein the cell is an MSC. 44. The method according to any one of claims 30 to 43,
And deriving offspring cells or tissues from the treated cells. 46. A cell or tissue obtained from the method of claim 44. Antibodies to SDC2 for use in human therapy. An antibody to SDC2 for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. 49. The method of claim 47,
Antibodies for use in immunosuppression. A polypeptide comprising or consisting of a fragment of SDC2, wherein the polypeptide has SDC2 activity and the fragment comprises no more than 150 amino acids of SEQ ID NO: 3. 50. The method of claim 49,
Wherein the fragment comprises no more than 120 amino acids of SEQ ID NO: 3. 50. The method of claim 49,
Wherein the fragment comprises no more than 100 amino acids of SEQ ID NO: 3. 52. The method according to any one of claims 49 to 51,
Wherein the fragment is an N-terminal fragment comprising at least amino acids 1 to 18 of SEQ ID NO: 3. 52. An antibody to a polypeptide of any of claims 49 to 52 for use in human therapy. 53. The method according to any one of claims 49 to 52,
(i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, or (iv) antibody to a polypeptide for use in wound healing. 55. The method of claim 54,
Antibodies for use in immunosuppression. Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; 57. The method of claim 56,
Lt; RTI ID = 0.0 &gt; SDC2-specific &lt; / RTI &gt; 57. The method of claim 56 or 57,
And comparing the SDC2 level to a pre-measured standard. 62. The method of any one of claims 56 to 58,
Wherein the method is performed to determine whether to treat a cell or tissue by a method according to any of the other methods of the present invention. Lt; RTI ID = 0.0 &gt; SDC2. &Lt; / RTI &gt; Use of SDC2 level in efficacy analysis of cell therapy products. 6. The method according to any one of claims 1 to 5,
A fragment of SEQ ID NO: 3 of at least 30 amino acids in length that retains at least 50% of the activity of native SDC2, or an SDC2 comprising said fragment. 63. The method of claim 62,
A fragment of SEQ ID NO: 3 with a length of at least 40 amino acids, or an SDC2 comprising said fragment. 63. The method of claim 62 or 63,
SDC2 comprising amino acids 1 to 18 of SEQ ID NO: 3. The method comprising administering to the patient an antibody to SDC2 or SDC2. A method of treating an inflammation in a patient, the method comprising administering to the patient an antibody against SDC2 or SDC2. A method of treating cancer in a patient, the method comprising administering to the patient an antibody to SDC2 or SDC2. A method of promoting wound healing in a patient, the method comprising administering to the patient an antibody against SDC2 or SDC2. antagonist to SDC2 for use in (i) immunosuppression, (ii) treatment of inflammation, (iii) treatment of cancer, (iv) wound healing, or (v) bone healing. 70. The method of claim 69,
A compound according to any one of claims 1 to 5, a vector according to claim 6 or 7, a compound according to any one of claims 8 to 19, a compound according to any one of claims 46 to 48 and An antibody according to any of claims 53 to 55, a polypeptide according to any one of claims 49 to 52, and an antagonist selected from SDC according to any one of claims 62 to 64 . 70. The method of claim 69 or 70,
Antagonists for use in immunosuppression. 70. The method of claim 69 or 70,
Antagonists for use in the treatment of inflammation. 70. The method of claim 69 or 70,
Antagonists for use in the treatment of cancer. 70. The method of claim 69 or 70,
Antagonist for use in wound healing. 70. The method of claim 69 or 70,
Antagonist for use in bone healing.

Images